Antibiotics have been seen for many years as “the answer” to the problem of bacterial infections. This attitude persisted until the development of the wide-ranging (and in some cases total) resistance to antibiotics seen within the last ten years. In many cases it is necessary to use expensive “drugs of last resort” (such as vancomycin for Staphylococcus aureus), which often require complex routes of administration and show toxic side effects, necessitating prolonged hospital treatment.
Even to these drugs, resistance is reaching worrying levels. It is now clear that bacteria can adapt to resist any antibiotic. Even the new generation drugs such as linezolid are already generating resistance [Mutnick et al (2003) An. Pharmacother. 37:769-774; Rahim et al (2003) Clin Infect Dis 36: E146-148], and it is clear from recent developments that resistance develops faster than new antibiotics can be produced, evaluated and processed through regulatory approvals.
A further disadvantage of antibiotic treatment is its lack of specificity. Antibiotics can kill a wide range of bacteria and this can lead to recolonisation of the body by inappropriate and often harmful bacteria. There is therefore need for antibacterial treatments that show specificity against particular bacterial species so that little resistance is induced in the normal flora.
The need for new forms of antibacterial therapy is well illustrated by the case of infection with the gram-negative aerobic bacterium Pseudomonas aeruginosa.
Pseudomonas aeruginosa is a serious opportunistic bacterial pathogen. Infections caused by Pseudomonas aeruginosa include:                Otitis externa and otitis media in dogs, ear infections which exemplify biofilm-based colonization of a body surface and which are common in inbred (pedigree) dogs;        Otitis externa of humans (“swimmers ear”) along with other ear infections and other topical infections of humans including Pseudomonas keratitis and Pseudomonas folliculitis;         Infection of burns and skin grafts in humans;        Hospital-acquired infections;        Lung infection in cystic fibrosis (CF) patients.        
0-15% of nosocomial (hospital acquired) infections are due to Pseudomonas Aeruginosa, with 2 million cases annually in the US alone. In some situations, the frequency is even higher. Of around 150,000 burn patients treated in US hospitals and burn centres per year, 26% have Pseudomonas aeruginosa infections. Pseudomonas aeruginosa is notorious for its resistance to antibiotics so infections caused by it can be difficult to treat. One of its natural habitats is soil, where it is exposed to organisms that produce antibiotics. This may well have led to the development of resistance mechanisms coded for both by genes on the chromosome and by transferable genetic elements known as plasmids. The properties of the P. aeruginosa outer membrane are important in conferring resistance. An additional resistance mechanism is its tendency to grow on available surfaces as complex layers known as biofilms [Donlan (2002) Emerging Infectious Diseases 8: 881-890, www.cdc.gov/ncidod/EID/vol8no9/02-0063.htm; Fletcher & Decho (2001) Biofilms in Encyclopaedia of Life Sciences, Nature Publishing, London; www.els.net] that are resistant to far higher concentrations of antibiotics than are required to kill individual cells [Chen et al (2002) Pseudomonas infection; www.emedicine.com/PED/topic2701.htm; Qarah et al (2001) Pseudomonas aeruginosa infections; www.emedicine.com/MED/topic1943.htm; Todar K. (2002) Todar's Online Textbook of Bacteriology: Pseudomonas aeruginosa; textbookofbacteriology.net/pseudomonas.html; Iglewski B. H (1996) Pseudomonas. Medical Microbiology 4th edition, S. Baron (ed.). University of Texas; gsbs.utmb.edu/microbook/ch027.htm]. The practical effect of this is demonstrated by infections in cystic fibrosis patients, virtually all of whom eventually become infected with a bacterial strain that cannot be eradicated by the use of antibiotics, even when the isolated strain may appear to be sensitive in the laboratory [Hoiby N (1998) Pseudomonas in cystic fibrosis: past, present, future. European Cystic Fibrosis Society Joseph Levy Memorial Lecture; www.ecfsoc.org/pa_review/nh_lect.html].
Pseudomonas aeruginosa expresses a range of genes (most notably the algC gene) which produce the extracellular components responsible for biofilm formation, which are often polysaccharide in nature (Friedman and Koller, Mol. Microbiol. (2004) 3, 675-690). Such biofilm formation is now known to be a characteristic of many important pathogenic bacteria contributing to increased resistance to antibiotics. Such biofilms may comprise more than one type of bacterium supported and surrounded by an excreted extracellular matrix and assist bacteria to colonise surfaces from marine reefs to teeth enamel. Biofilms allow bacteria to attach to surfaces and to attain population densities which would otherwise be unsupportable. They impart increased resistance to not only antibiotics but many environmental stresses including toxins such as heavy metals, bleaches and other cleaning agents. It was previously thought that contribution of biofilm formation to antibiotic resistance was primarily a physical process arising from limitation of diffusion, but more recent evidence has shown that some biofilms appear to have specific abilities to trap antibiotics (Mah et al., Nature (2003) 426, 306-310). It is known that bacteria within biofilms can be 100 to 1000 times more resistant to antibiotics than the same strain of bacteria growing in single-celled (“planktonic”) forms. This increased resistance means that bacteria that are apparently sensitive to antibiotics in a laboratory test may be resistant to therapy in a clinical setting. Even if some are cleared, biofilms may provide resistant reservoirs permitting rapid colonisation once antibiotics are no longer present. It is clear therefore that biofilms are major factors in many human diseases.
Chemical treatments are unsuited to use against biofilms since this is precisely what they have evolved to counter and many surfaces where biofilms aid bacterial pathogenesis are poorly suited to rigorous abrasion. Physical abrasion does provide a means to disrupt biofilms. However, many surfaces where biofilms aid bacterial pathogenesis are poorly suited to rigorous abrasion. For example, the surfaces of wounds or burns are extremely sensitive and delicate. Even where abrasion is both suitable and in routine use, clearing of biofilms is limited. Oral plaque on the surface of teeth is a biofilm and is partially cleared by regular brushing. However, bacteria are maintained on unbrushed surfaces (for example in the gaps between teeth) and can recolonise cleared surfaces both rapidly and effectively. From this, it is clear that existing approaches to clearing biofilms are of limited efficacy.
In addition to the biofilm problem, only a few antibiotics in any case are capable of effective action against Pseudomonas aeruginosa, including fluoroquinolones, gentamicin and imipenem, and even these antibiotics are not effective against all strains. Multidrug resistance is common and increasing [Friedland I et al (2003). Diagnostic Microbiology and Infectious Disease 45:245-50; Henwood et al (2001). Journal of Antimicrobial Chemotherapy 47: 789-799]. The U.S. National Nosocomial Infections Surveillance System Report of June 1999 [Gerberding Jet al (2001). National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992-June 2001, issued August 2001. U.S. Department of Health and Human Services, Atlanta, www.cdc.gov/ncidod/hip/NNIS/2001nnis_report.PDF] states that antibiotic resistance of Pseudomonas aeruginosa isolated from nosocomial infections in ICU patients in 1999 had increased over the 1994-98 period for all classes of antibiotics studied. There is therefore a demonstrated need for new approaches to the control of Pseudomonas aeruginosa infection. The inventors in this instance have addressed this problem through use of new bacteriophage-based therapies.
Bacteriophages (often known simply as “phages”) are viruses that grow within bacteria. The name translates as “eaters of bacteria” and reflects the fact that as they grow most bacteriophages kill the bacterial host as the next generation of bacteriophages is released. Early work with bacteriophages was hindered by many factors, one of which was the widespread belief that there was only one type of bacteriophage, a non-specific virus that killed all bacteria. In fact, the host range of bacteriophages (the spectrum of bacteria they are capable of infecting) is often very specific. This specificity may be considered a therapeutic strength as populations of bacteriophages can be selected to specifically eliminate only the target bacteria. Antibiotics, on the other hand, kill a wide range of bacteria and their use can consequently lead to disruption of the normal flora, leading to recolonisation of the body by inappropriate and often harmful bacteria.
Despite the therapeutic advantages afforded by the host specificity of bacteriophages, this characteristic has the disadvantage that it can be difficult to achieve breadth of coverage of target strains. For this reason, there has been interest in finding combinations of bacteriophages having broad targeting capability in relation to particular types of bacterial infection (see for example Pirsi, The Lancet (2000) 355,1418)
The inventors in this instance have established a combination of bacteriophages consisting of six bacteriophages each with a different strain specificity against Pseudomonas aerguinosa and which is particularly suitable for broad targeting of P. aerguinosa infections, especially, for example, canine ear infections. The combination was found to be capable of destroying 90% of P. aeruginosa strains sampled from canine otitis externa and other canine ear infections. Furthermore, they have established that such a phage combination may be employed synergistically with antibiotic treatment to gain improved efficacy. As a consequence, it is now extrapolated that combined phage/antibiotic therapy represents a new general advantageous approach for tackling bacterial infections characterised by biofilm formation.
Phage and antibiotic therapy have previously been used together in Eastern Europe (see for example Bradbury, The Lancet (February 2004) 363, 624-625), but there was no specific relation to biofilm formation. Additionally, there have been suggestions that antibiotics can have adverse effect on use of bacteriophage therapy since bacteriophages use bacterial metabolism to replicate and this is inhibited by antibiotics (Payne and Janssen, Clinical Pharmacokinetics (2002) 42, 315-325).